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Preparation method for replication and transcription activator (Rta) protein and application of Rta protein to nasopharynx cancer detection reagent

A technology of nasopharyngeal carcinoma and protein, applied in the field of serological detection of nasopharyngeal carcinoma, can solve the problems of unsatisfactory sensitivity, false positive results, interference detection, etc., and achieve the effect of improving sensitivity and specificity

Inactive Publication Date: 2012-10-03
同昕生物技术(北京)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the diagnostic effect of this solution is higher than the international standard, there is still a big gap with the accuracy of pathological examination of nasopharyngeal carcinoma biopsy, which makes the kit unable to play the role of Rta molecule in clinical diagnosis and anticancer treatment monitoring. The inherent high sensitivity and high specificity indicators of the target should be used for antibody detection or as a vaccine, there are problems of missed detection or low titer, prone to cross-reaction, interference detection and false positive results
Simultaneously in the Chinese patent ZL200610113403, only selected part of the reading frame of BRLF1 gene as the antigen for antibody detection, but the sensitivity is unsatisfactory

Method used

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  • Preparation method for replication and transcription activator (Rta) protein and application of Rta protein to nasopharynx cancer detection reagent
  • Preparation method for replication and transcription activator (Rta) protein and application of Rta protein to nasopharynx cancer detection reagent
  • Preparation method for replication and transcription activator (Rta) protein and application of Rta protein to nasopharynx cancer detection reagent

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Experimental program
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Effect test

Embodiment 1

[0028] 1. Design and synthesis of primers

[0029] The PCR primer sequences designed according to the known BRLF1 sequence (GenBank number gi:94734074) are, Rta-full-lowprimer: ccTCTAGAaaataagctggtgtc (sequence 2 in the sequence listing) Rta-full-upprimer: gcGAATTCatgaggcctaaaaaggatg (sequence 3 in the sequence listing), with There are EcoRI, XbaI enzyme cutting sites, synthesized by Shanghai Sangong.

[0030] The sequence of the expressed protein corresponding to the BRLF1 sequence is GenBank gi:94734074.

[0031] 2. Obtain the cDNA of the BRLF1 gene and clone it into the vector:

[0032] B95-8 cells (ATCC Number: CRL- 10624 TM The EBV in ) entered the lytic phase, and the cDNA of BRLF1 gene was obtained by RT-PCR method, and cloned into pEASY-blunt-simple (Beijing Quanshijin Biotechnology Co., Ltd.) vector, transfected into Escherichia coli competent cells, challenged After the single clones were cultured, the plasmids were extracted and bidirectional assays were perform...

Embodiment 2

[0043] Embodiment 2, establish human serum Rta-IgG detection method with the Rta antigen of eukaryotic expression

[0044] Using the Rta protein prepared in Example 1 as an antigen, an indirect method for detecting the Rta-IgG antibody in human serum was established. The Rta antibody in the plasma sample is combined, and then HRP-labeled goat anti-human IgG is added to bind it, and then TMB substrate is added to develop the color, and then the reaction is terminated with the stop solution. The absorbance (A value) was detected by a microplate reader. The value of Abs is directly proportional to the concentration of Rta-IgG antibody.

[0045] Proceed as follows:

[0046] 1. Preparation of coated microtiter plates:

[0047] Dilute the purified eukaryotic expression Rta antigen to 0.1ng / ml (0.05-0.5ng / ml) with coating buffer (pH9.6, 0.05mol / L carbonate buffer) and add to the microtiter plate 100ul per well, react at 37°C for 2 hours, shake off the coating solution, wash with ...

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Abstract

The invention discloses a preparation method for a replication and transcription activator (Rta) protein and the application of the Rta protein to a nasopharynx cancer detection reagent and relates to a medical diagnosis reagent. The preparation method disclosed by the invention comprises the following steps of: 1, constructing a recombinant expression vector by taking a BRLF1 full-length gene as an exogenous gene; 2, transfecting: transfecting the recombinant expression vector into an eukaryotic expression system to obtain a positive transfected cell; and 3, expressing and purifying: culturing the positive transfected cell so as to enable the positive transfected cell to express an interest protein, and separating and purifying the interest protein, wherein the eukaryotic expression system refers to a Chinese hamster ovary (CHO) cell. The Rta protein prepared by the method disclosed by the invention is used for detecting nasopharynx cancer; the sensitivity of the Rta protein is 96 percent (288 / 300), and the specificity of the Rta protein is 96.7 percent (290 / 300). The sensitivity and the specificity are superior to those of antigens respectively prepared by a prokaryotic expression system and a pichia expression system, and the sensitivity and the specificity on clinical early diagnosis on the nasopharynx cancer are greatly improved.

Description

technical field [0001] The present invention relates to medical diagnostic reagents, specifically, the present invention relates to a method for expressing BRLF1 full-length gene using a eukaryotic expression system to obtain a purified expression product Rta, and the obtained Rta protein is used in the serological detection of nasopharyngeal carcinoma . Background technique [0002] Epstein-Barr virus is a gamma herpes virus that infects approximately 95% of adults worldwide. Epstein-Barr virus infection can be divided into two states: latent infection period and cleavage replication period. After the initial infection, the virus can establish a lifelong latent infection in the host, and persistent EB virus cleavage and replication state infection can lead to a series of human malignant diseases. Tumors (Rickinson AB, Lee SP, Steven NM. Cytotoxic T lymphocyte responses to Epstein-Barr virus. Curr Opin Immunol. 1996 Aug;8(4):492-7). The BRLF1 gene is located in ORF50 of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/38C07K14/05G01N33/68G01N33/543
Inventor 李全焦守恕吴凡
Owner 同昕生物技术(北京)有限公司
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